Millonig R, Salvo H, Aebi U
M. E. Müller Institute for High Resolution Electron Microscopy, University of Basel, Switzerland.
J Cell Biol. 1988 Mar;106(3):785-96. doi: 10.1083/jcb.106.3.785.
We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.
我们使用了N,N'-1,4-亚苯基双马来酰亚胺，一种双功能巯基交联试剂，来探究肌动蛋白聚合成丝早期阶段的寡聚状态。我们证明，在多种离子条件下，球状单体肌动蛋白（G-肌动蛋白）聚合的第一步之一是相当一部分G-肌动蛋白单体库发生二聚化。随着聚合反应的进行，这种初始二聚体（通过SDS-PAGE显示表观分子量为86 kD的“较低”二聚体[LD]）的产量降低，而形成了一种肌动蛋白丝二聚体（通过SDS-PAGE显示表观分子量为115 kD的“较高”二聚体[UD]，已得到表征[埃尔津加，M.，和J. J. 费兰。1984年。美国国家科学院院刊。81:6599 - 6602]）。从LD到UD的这种转变与通过N-(1-芘基)碘乙酰胺荧光增强和电子显微镜检测到的丝的形成同时发生。分离的交联LD不会形成丝，而分离的交联UD在典型的丝形成条件下会组装成与由未修饰的G-肌动蛋白聚合而成的丝难以区分的丝。交联LD的存在不会影响肌动蛋白单体的聚合动力学，而交联UD则以浓度依赖的方式缩短聚合反应的“滞后阶段”。几条相互印证的证据表明，尽管LD是早期聚合过程中形成的一种重要的寡聚体，但它与定义成熟肌动蛋白丝的螺旋对称性不相容；然而，它可能代表在副晶阵列或丝束中发现的丝间二聚体。此外，LD与各种类型的结晶肌动蛋白阵列共有的晶胞结构和对称性（埃比，U.，W. E. 福勒，G. 伊森伯格，T. D. 波拉德，和P. R. 史密斯。1981年。细胞生物学杂志。91:340 - 351）相容，并且可能代表一种突变β-肌动蛋白（利维特，J.，G. 布沙尔，T. 卡库纳加，H. 滨田，T. 平川，D. 戈德曼，和C. 梅里尔。1982年。细胞。28:259 - 268）在聚合条件下停滞的主要结构状态。
